AIM: Activating mutations in the epidermal growth factor receptor (EGFR) are predominantly detected in pulmonary adenocarcinoma and have been reported in small cell lung cancer (SCLC) for decades. This retrospective single-center study aimed to determine the frequency and types of EGFR mutations in SCLC in Taiwan. METHODS: This study comprises a consecutive cohort of 161 patients histologically diagnosed with SCLC between January 1992 and August 2014 at the Department of Pathology in Keelung Chang Gung Memorial Hospital, Taiwan. Archived formalin-fixed paraffin-embedded sections from 71 patients were eligible for molecular analysis. EGFR mutation analysis was performed using a fully-automated IdyllaTM EGFR Mutation Test and confirmed a comparable result through Qiagen Therascreen® EGFR RGQ PCR. In addition, EGFR gene copy number was assessed in EGFR-mutated tumors by fluorescence in situ hybridization (FISH). RESULTS: Mutational status of the EGFR gene was successfully analyzed in 63 specimens by both IdyllaTM and Qiagen platforms. Both methods detected L858R point mutation in exon 21 in an 81-year-old female and a 47-year-old male non-smoker. Both tumors show no concurrent EGFR gene amplification. The overall agreement between results obtained with the IdyllaTM EGFR Mutation Test and Qiagen Therascreen® EGFR RGQ PCR was 100% Conclusions. Our results showed that EGFR mutation is a rare mutation type in a consecutive series of de novo SCLC. Furthermore, the performance of IdyllaTM EGFR Mutation Test and Qiagen Therascreen® EGFR RGQ PCR on archived paraffin sections of limited quantities is available with the high agreement of results.

• MeSH
• erbB receptory genetika   MeSH
• formaldehyd MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• malobuněčný karcinom plic * genetika   MeSH
• mutace MeSH
• mutační analýza DNA metody   MeSH
• nádory plic * diagnóza   MeSH
• nemalobuněčný karcinom plic * diagnóza   MeSH
• parafín MeSH
• retrospektivní studie MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH

Kryptokokální meningitida (KM) je potenciálně fatální onemocnění. Popisujeme úspěšnou léčbu pomocí Om­maya rezervoáru a intratekální injekce amfotericinu B ke zvládnutí refrakterní cerebrální kryptokokózy způsobené Cryptococcus gattii AFLP4/VGI. U 63letého pa­cienta s hypertermií a bolestmi hlavy v trvání 2 týdnů byla dia­gnostikována KM. Byla zahájena kombinovaná léčba amfotericinem B a 5-flucytosinem a následována léčbou flukonazolem. Po 7 měsících byl refrakterní k tradiční léčbě KM a byla zvolena záchran­ná léčba pomocí Om­maya rezervoáru a intratékální injekce amfotericinu B. Neurologické příznaky postupně odezněly bez zřejmého relapsu během 12 měsíců sledování. Izolát byl přešetřen kultivací na médiu s kanavaninem, glycinem a bromthymolovou modří a molekulární typizací pomocí URA5 byl identifikován Cryptococcus gattii AFLP4/VGI. Om­maya rezervoár lze doporučit jako alternativní léčbu KM vyvolané Cryptococcus gattii AFLP4/VGI, která špatně odpovídá na standardní léčebné režimy.

Cryptococcal meningitis (CM) is a potentially fatal disease. We report successful treatment with the Ommaya reservoir and intrathecal injection of amphotericin B for the control of refractory cerebral cryptococcosis due to Cryptococcus gattii AFLP4/VGI. A 63-year-old male patient presented with a 2-week history of fever and headache, and was he diagnosed with CM. Combined amphotericin B and 5-flucytosine treatment was initiated and followed by fluconazole therapy. After 7 months, he was refractory to traditional CM treatment and received salvage therapy with an Ommaya reservoir and intrathecal injection of amphotericin B. His neurological symptoms recovered gradually with no evidence of relapse during 12-month follow-up. The isolate was re-identified by culturing in canavanine-glycine-bromothymol Blue media and by molecular typing using URA5 as Cryptococcus gattii AFLP4/VGI. The Ommaya reservoir could serve as an alternative treatment for Cryptococcus gattii AFLP4/VGI-induced CM, which responds poorly to standard regimens.

• Klíčová slova
• Ommaya rezervoár,
• MeSH
• amfotericin B terapeutické užití   MeSH
• antifungální látky terapeutické užití   MeSH
• Cryptococcus gattii izolace a purifikace   MeSH
• flucytosin terapeutické užití   MeSH
• flukonazol MeSH
• hydrocefalus etiologie   MeSH
• kombinovaná farmakoterapie MeSH
• kryptokoková meningitida * diagnóza   farmakoterapie   patofyziologie   MeSH
• léková rezistence MeSH
• lékové transportní systémy MeSH
• lidé středního věku MeSH
• lidé MeSH
• mozkové komory * účinky léků   MeSH
• recidiva MeSH
• spinální injekce MeSH
• zaváděcí katétry * MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH
• práce podpořená grantem MeSH

In plants, membrane compartmentalization requires vesicle trafficking for communication among distinct organelles. Membrane proteins involved in vesicle trafficking are highly dynamic and can respond rapidly to changes in the environment and to cellular signals. Capturing their localization and dynamics is thus essential for understanding the mechanisms underlying vesicular trafficking pathways. Quantitative mass spectrometry and imaging approaches allow a system-wide dissection of the vesicular proteome, the characterization of ligand-receptor pairs and the determination of secretory, endocytic, recycling and vacuolar trafficking pathways. In this review, we highlight major proteomics and imaging methods employed to determine the location, distribution and abundance of proteins within given trafficking routes. We focus in particular on methodologies for the elucidation of vesicle protein dynamics and interactions and their connections to downstream signalling outputs. Finally, we assess their biological applications in exploring different cellular and subcellular processes.

• MeSH
• biologický transport MeSH
• endocytóza MeSH
• hmotnostní spektrometrie metody   MeSH
• proteom * analýza   metabolismus   MeSH
• proteomika * metody   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

• MeSH
• gynekologická onemocnění MeSH
• komplementární terapie MeSH
• tradiční čínská medicína MeSH
• Publikační typ
• příručky MeSH

• Konspekt
• Gynekologie. Porodnictví
• NLK Obory
• gynekologie a porodnictví
• alternativní lékařství

Feedstock type influences bacterial and methanogenic communities in anaerobic digestion. These two communities work tightly to maintain the stability of anaerobic digestion. How to quick report the changes of microbial community structure especially methanogenesis is the key issue for optimizing anaerobic digestion process. In this study, 13C isotope fractionations of CH4 and CO2 in biogas and microbial community composition were analyzed in 5 different feedstocks. Our results showed that grass silage, maize silage and swine manure fed reactors had similar δ 13C values and methanogenic community composition, dominated by Methanosarcinaceae. The lowest δ 13CH4 values were detected in straw and chicken manure fed reactors, reflecting reduced microbial degradation of material or the presence of toxic components in these feedstocks. The straw fed bioreactor lead to low δ 13CH4 values, probably reflecting relatively high levels of the syntrophic acetate oxidizing bacteria, Synergistaceae and Syntrophaceae, which might work collectively with hydrogenotrophic methanogens, resulting in the low δ 13CH4 values in this bioreactor. Significantly, all core microbes in the 5 different feedstock fed bioreactors were either Clostridia species or related to the Synergistaceae (syntrophic acetate oxidizing bacteria).

• MeSH
• anaerobióza MeSH
• biopaliva * MeSH
• bioreaktory MeSH
• hnůj MeSH
• izotopy MeSH
• methan MeSH
• mikrobiota * MeSH
• prasata MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The aim of this study was to investigate how the microbial community structure adapts during the start-up phase and how the 13C fractionation of biogas reflects the microbial population dynamics in two parallel swine manure-fed anaerobic digesters. Two swine manure-fed reactors for the start-up of continuously stirred tank reactors at mesophilic condition were evaluated. Changes in community structure were monitored using 16S rRNA high-throughput sequencing to measure the abundance of fermenting bacteria and methanogens. Digesters with relatively stable Methanosarcinaceae started up successfully and contained high gas production and low levels of propionate. In contrast, the digester that experienced a difficult start-up period had reduced Methanosarcinaceae along with accumulated propionate and low gas production. Specific gas production, specific methane production, and 13C fractionation of biogas were influenced significantly by Methanosarcinaceae, Methanobacteriaceae, and Clostridiaceae, indicating that the 13C fractionation of biogas had significant potential to reflect microbial population changes and digester performance during the start-up period.

• MeSH
• anaerobióza MeSH
• Bacteria MeSH
• biopaliva MeSH
• bioreaktory mikrobiologie   MeSH
• chemická frakcionace MeSH
• fermentace MeSH
• hnůj mikrobiologie   MeSH
• prasata MeSH
• RNA ribozomální 16S genetika   MeSH
• uhlík chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Cíl Přestože již bylo prokázáno, že inhibitory tyrosinkináz (tyrosine kinase inhibitor, TKI) receptoru epidermálního růstového faktoru (epidermal growth factor receptor, EGFR) jsou u pacientů s adenokarcinomem plic s aktivující mutací EGFR účinnější než u pacientů s tímto genem divokého typu, je v první skupině přítomno zhruba 30 % pacientů, kteří na TKI neodpovídají. Molekulární základ této zásadní heterogenity odpovědí není znám. Naším cílem bylo najít molekulární aberace, které na celogenomové úrovni přispívají k progresi onemocnění, a určit prognostickou signaturu specifi ckou pro pacienty s aktivující mutací EGFR. Pacienti a metody Nejprve jsme pomocí microarray pro vysokodenzitní komparativní genomovou hybridizaci (high-density array comparative genomic hybridization) v souboru 138 tkání adenokarcinomu prozkoumali molekulární rozdíly mezi nádory s aktivující mutací EGFR a nádory s genem divokého typu. Potom jsme u jiné nezávislé skupiny 114 pacientů validovali klinický význam alterací počtu kopií (copy-number alteration, CNA) pro predikci celkového přežití a přežití bez známek onemocnění (disease-free survival, DFS). V posledním kroku jsme u 23 pacientů s mutací EGFR léčených EGFR TKI zkoumali spojení mezi CNA a odpovědí na EGFR-TKI. Výsledky Určili jsme chromosomové oblasti s rozdíly v CNA mezi nádory s aktivující mutací EGFR a nádory divokého typu a zjistili jsme přítomnost vysoké míry shlukování aberací na chromosomu 7p. Klastr šesti reprezentativních genů na chromosomu 7p předpovídal celkové přežití a přežití bez známek onemocnění pacientů s aktivující mutací EGFR, ale ne u pacientů s genem divokého typu. Důležité je zjištění, že současná přítomnost většího počtu genů se zvýšenou hodnotou CNA v tomto klastru u pacientů s aktivující mutací EGFR korelovala s méně příznivou odpovědí na EGFR-TKI. Závěr Naše výsledky významným způsobem přispívají k objasnění příčin heterogenních odpovědí pacientů s aktivující mutací EGFR na léčbu EGFR-TKI. Mohly by přispět ke zlepšení léčby pacientů v této populaci.

PURPOSE: Although epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been proven more effective for patients with lung adenocarcinoma with EGFR-activating mutation rather than wild type, the former group still includes approximately 30% nonresponders. The molecular basis of this substantial response heterogeneity is unknown. Our purpose was to seek molecular aberrations contributing to disease progression at the genome-wide level and identify the prognostic signature unique to patients with EGFR-activating mutation. PATIENTS AND METHODS: We first investigated the molecular differences between tumors with EGFR-activating mutation and wild-type tumors by conducting high-density array comparative genomic hybridization on a collection of 138 adenocarcinoma tissues. We then used an independent group of 114 patients to validate the clinical relevance of copy-number alterations (CNAs) in predicting overall and disease-free survival. Finally, focusing on 23 patients with EGFR mutation receiving EGFR-TKI treatment, we investigated the association between CNAs and response to EGFR-TKIs. RESULTS: We identified chromosome regions with differential CNAs between tumors with EGFR-activating mutation and wild-type tumors and found the aberration sites to cluster highly on chromosome 7p. A cluster of six representative chromosome 7p genes predicted overall and disease-free survival for patients with EGFR-activating mutation but not for those with wild type. Importantly, simultaneous presence of more genes with increased CNAs in this cluster correlated with less favorable response to EGFR-TKIs in patients with EGFR-activating mutation. CONCLUSION: Our results shed light on why responses to EGFR-TKIs are heterogeneous among patients with EGFR-activating mutation. They may lead to better patient management in this population.

• MeSH
• adenokarcinom farmakoterapie   genetika   MeSH
• chromozomální aberace MeSH
• DNA nádorová genetika   MeSH
• erbB receptory genetika   MeSH
• financování organizované MeSH
• genom lidský MeSH
• genová dávka MeSH
• inhibitory proteinkinas terapeutické užití   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lidské chromozomy, pár 7 genetika   MeSH
• míra přežití MeSH
• mutace genetika   MeSH
• mutační analýza DNA MeSH
• nádory plic farmakoterapie   genetika   MeSH
• následné studie MeSH
• nemalobuněčný karcinom plic farmakoterapie   genetika   MeSH
• polymerázová řetězová reakce MeSH
• prognóza MeSH
• senioři MeSH
• srovnávací genomová hybridizace MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH

The aim of this study was to evaluate the effect of raw (RawBC) and iron (Fe)-modified biochar (FeBC) derived from Platanus orientalis Linn branches on the plant growth, enzyme activity, and bioavailability and uptake of As, Cd, and Pb by rice in a paddy soil with continuously flooded (CF) or alternately wet and dry (AWD) irrigation in a pot experiment. Application of RawBC (3%, w/w) significantly increased soil pH, while FeBC decreased it. The FeBC was more effective in reducing As and Pb bioavailability, particularly under the AWD water regime, while RawBC was more conducive in reducing Cd bioavailability under the CF water regime. The FeBC decreased As concentration, but increased concentrations of Cd and Pb in the straw and brown rice, as compared to the untreated soil. Soil catalase and urease activities were enhanced by RawBC, but decreased by FeBC treatment. The FeBC increased the grain yield by 60% and 32% in CF and AWD treatments, respectively. The FeBC can be recommended for immobilization of As in paddy soils, but a potential human health risk from Cd and Pb in FeBC-treated soils should be considered due to increased uptake and translocation of the metals to brown rice.

• MeSH
• arsen * MeSH
• dřevěné a živočišné uhlí MeSH
• kadmium analýza   MeSH
• látky znečišťující půdu * analýza   MeSH
• lidé MeSH
• olovo MeSH
• půda MeSH
• rýže (rod) * MeSH
• voda MeSH
• zásobování vodou MeSH
• železo MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Alpers' syndrome is an early-onset neurodegenerative disorder usually caused by biallelic pathogenic variants in the gene encoding the catalytic subunit of polymerase-gamma (POLG), which is essential for mitochondrial DNA (mtDNA) replication. The disease is progressive, incurable, and inevitably it leads to death from drug-resistant status epilepticus. The neurological features of Alpers' syndrome are intractable epilepsy and developmental regression, with no effective treatment; the underlying mechanisms are still elusive, partially due to lack of good experimental models. Here, we generated the patient derived induced pluripotent stem cells (iPSCs) from one Alpers' patient carrying the compound heterozygous mutations of A467T (c.1399G>A) and P589L (c.1766C>T), and further differentiated them into cortical organoids and neural stem cells (NSCs) for mechanistic studies of neural dysfunction in Alpers' syndrome. Patient cortical organoids exhibited a phenotype that faithfully replicated the molecular changes found in patient postmortem brain tissue, as evidenced by cortical neuronal loss and depletion of mtDNA and complex I (CI). Patient NSCs showed mitochondrial dysfunction leading to ROS overproduction and downregulation of the NADH pathway. More importantly, the NAD+ precursor nicotinamide riboside (NR) significantly ameliorated mitochondrial defects in patient brain organoids. Our findings demonstrate that the iPSC model and brain organoids are good in vitro models of Alpers' disease; this first-in-its-kind stem cell platform for Alpers' syndrome enables therapeutic exploration and has identified NR as a viable drug candidate for Alpers' disease and, potentially, other mitochondrial diseases with similar causes.

• MeSH
• DNA polymeráza gama MeSH
• indukované pluripotentní kmenové buňky * MeSH
• lidé MeSH
• mitochondriální DNA genetika   MeSH
• mitochondriální nemoci * MeSH
• mutace MeSH
• NAD genetika   MeSH
• niacinamid analogy a deriváty   MeSH
• pyridinové sloučeniny * MeSH
• Schilderova difuzní cerebroskleróza * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Proteases are typical key enzymes that hydrolyze proteins into amino acids and peptides. Numerous proteases have been studied, but the discovery of metagenome-derived proteases is still significant for both commercial applications and basic research. An unexplored protease gene sep1A was identified by function-based screening from a plasmid metagenomic library derived from uncultured contaminated agricultural soil microorganisms. The putative protease gene was subcloned into pET-32a (+) vector and overexpressed in E. coli BL21(DE3) pLysS, then the recombinant protein was purified to homogeneity. The detailed biochemical characterization of the Sep1A protein was performed, including its molecular characterization, specific activity, pH-activity profile, metal ion-activity profile, and enzyme kinetic assays. Furthermore, the protein engineering approach of random mutagenesis via error-prone PCR was applied on the original Sep1A protein. Biochemical characterization demonstrated that the purified recombinant Ep48 protein could hydrolyze casein. Compared with the original Sep1A protein, the best variant of Ep48 in the random mutagenesis library, with the Gln307Leu and Asp391Gly changes, exhibited 2.62-fold activity at the optimal reaction conditions of 50 °C and pH 9.0. These results are the first step toward a better understanding of the properties of Sep1A protein. Protein engineering with error-prone PCR paves the way toward the metagenome-derived genes for biotechnological applications.

• MeSH
• Bacteria chemie   enzymologie   genetika   izolace a purifikace   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• genová knihovna MeSH
• kinetika MeSH
• klonování DNA MeSH
• metagenom MeSH
• molekulární sekvence - údaje MeSH
• mutageneze MeSH
• proteasy chemie   genetika   metabolismus   MeSH
• půdní mikrobiologie * MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• stabilita enzymů MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH